There are over 100 different forms of arthritis. The most common form is osteoarthritis (degenerative joint disease). Osteoarthritis is most commonly the result of a trauma or of an infection of the joint, albeit that there are also not readily identifiable causes. The latter are often collectively referred to as “age-related osteoarthritis.” Other forms of arthritis are, for example, rheumatoid arthritis, psoriatic arthritis, and related autoimmune diseases.
The major complaint by individuals who have arthritis is joint pain. Pain is often constant and may be localized to the joint affected. The pain from arthritis is often the result of damage to the joint or the result of the inflammation that occurs around the joint. Other complaints are pain as a result of muscle strains caused by forceful movements against stiff, painful joints and fatigue.
The diagnosis of patients with joint-associated pain is not easy as the complaints are typically vague and can be attributed to a variety of different causes, some of which are not arthritis. Indeed, many patients that first see a doctor with joint pain-associated complaints go into remission and don't develop a chronic form of arthritis, whereas a significant minority progress to develop Rheumatoid Arthritis (RA). It is clear that individuals that go into remission spontaneously do not need to receive treatment, whereas individuals that progress would benefit considerably from early treatment. To discriminate between the respective groups, the field has developed a series of tests with which the diagnosis of arthritis can be made with more certainty. Such tests presently involve the screening of tissue samples and/or body fluid samples for the presence of arthritis indicators therein. Such indicators are, among others, determination of “chronic” inflammation indicators such as, for instance, certain chemokines, cytokines and other immune cell signaling factors; the determination of “accumulation of” active immune cells in joints, and/or the presence of the certain factors in the blood, the most notable of which is rheumatoid factor. Recently, tests directed toward the detection of citrullinated protein or peptides or antibodies specific for such citrullinated protein or peptide have been developed as a useful tool for such tests. The availability of such tests has greatly improved the diagnosis of individuals suspected of having a form of arthritis. These tests also aid the clinician in giving a more accurate prognosis for the future development of the disease to individuals suffering from arthritis. However, in spite of these developments, the diagnosis of arthritis or individuals at risk thereof still leaves much to be desired.
For example, in the Netherlands, the recommendation to diagnose RA is based on a probability score generated by the ACR/EULAR 2010 criteria. This criteria combines clinical features such as involvement of type and number of joints, presence or absence of the serological factors, rheumatoid factor and anti-CCP antibodies, presence or absence of acute phase proteins such as CRP, and duration of complaints. Patients that are diagnosed with RA according to this protocol need to have more than six points. The fact that only the clinical involvement and duration of complaints are sufficient for a positive diagnosis indicates that the arthritis population is very heterogeneous and, in fact, often a wrong diagnosis is made.
In the Netherlands and in other countries, tests for the typing of arthritis or diagnoses of an individual at risk of developing arthritis presently include tests for the detection of antibodies specific for citrullinated protein or peptide in samples of body fluids of such patients. These tests have led to the insight that arthritis patients can be classified on the basis of positivity for antibodies directed toward citrullinated proteins (ACPA). The identification of ACPA has had an important impact on the understanding of RA.(1) Major differences have been observed when comparing ACPA-positive vs. ACPA-negative RA patients regarding genetic and environmental risk factors,(2) progression,(3) remission,(4) and response to treatment.(5) Over the recent years, much more insight has been gained into the occurrence and etiopathology of ACPA-positive RA. However, much less information is available on ACPA-negative RA. In part, this is because it is relatively difficult to identify or even subgroup these individuals as no good assays are currently available. Interestingly, rituximab treatment has been reported to also be beneficial in patients negative for RF and ACPA.(6, 7) 
The post-translational modification of arginine residues into citrulline residues by the PAD enzymes is the essential step to generate antigens for ACPA.(1) Under physiological circumstances, this citrullination is important in tissues like hair and skin to generate layers of tissue that are not-well connected.(8) Also, in the nucleus, citrullination plays a role in epigenetic regulation(9) and condensation of chromatin, which is important, both in translation(8) and in host defense against pathogens.(10) Under pathological conditions where cell death may overwhelm the phagocytic capacity, necrotic cell death may release PAD into the extracellular space, where higher calcium concentrations now also allow other host molecules to become citrullinated.(8) Since many of these molecules will be presented to the immune system as non-self, it can induce an antibody response in some individuals. Citrulline highly resembles (FIG. 1) another post-translationally modified amino acid called homocitrulline.(11) Homocitrulline is only one carbon longer, but similar in structure.(11) Homocitrulline is generated from a Lysine residue following an attack from cyanate, which exists in the body in equilibrium with urea. Under physiological conditions, the urea concentration may be too low to allow extensive carbamylation (the process of changing lysine to homocitrulline). In conditions of renal failure, the urea concentration increases and carbamylation can be readily detected. However, most carbamylation is taking place during inflammation when myeloperoxidase (MPO) is released from neutrophils.(12) This enzyme strongly shifts the equilibrium of urea toward cyanate, now allowing more carbamylation to occur.(13) It has been shown recently that homocitrulline-containing proteins are present in the RA joint and that this may affect T-cell triggering and autoantibody formation in animal models.(11, 14) Although highly similar, carbamylation differs from citrullination as, next to their structural difference, lysine and not arginine is modified. Therefore, homocitrulline will, by definition, be located at other positions in proteins than citrulline.